Morpho-physiological and biochemical changes was carried out for 16 germplasm lines of Urdbean against disease powdery mildew caused by Erysiphe polygoni, where KUP-34 recorded as significantly high in leaf thickness, phenol content, trichome density, and lowest in total sugars, stomatal size, reducing sugars and non-reducing sugars compared to highly susceptible genotype LBG-623.
Trang 1Original Research Article https://doi.org/10.20546/ijcmas.2020.907.215
Erysiphe polygoni Induced Morpho-physiological and Biochemical Changes
in Blackgram (Vigna mungo)
Tulasi Korra 1 * and V Manoj Kumar 2
1
Department of Mycology and Plant Pathology, Banaras Hindu University, Varanasi, India
2
Department of Plant Pathology, Acharya N Ranga Agricultural University, Bapatla, LAM,
Guntur, India
*Corresponding author
A B S T R A C T
Introduction
Black gram is essential seed crop and the best
source of phosphoric acid in the pluses and is
an significant dietary protein level (Duffus
and Slaughter, 1980) It’s seed has the highest
protein level (25g/100g) with other
aminoacids and minerals such as potassium,
calcium, iron, niacin, thamine and riboflavin
Urdbean is an mini-fertilizer factory, which
rehabilitates soil fecundity by raising
atmospheric nitrogen, generates an equivalent
of ‘N’ of approximately 22 kg per hectare
(Rachie and Roberts, 1974) Blackgram
genesis from Central Asia and currently found
in tropical and subtropical areas around
Africa, Asia, and Madagascar (Arora et al.,
1989) It was domesticated in the neighbouring regions of South Asia Globally, the production of black gram from the main producer countries like India, Myanmar or Thailand amounts to around 8.5 million tonnes
The cultivated in an area of 3.56 Mt and 655 kg/ha was such as a third important pluses crop in a India (Department Of Farming and Co-Operation, The Government of India, 2018) in region of 5.44Mh, with output
ISSN: 2319-7706 Volume 9 Number 7 (2020)
Journal homepage: http://www.ijcmas.com
Morpho-physiological and biochemical changes was carried out for 16
germplasm lines of Urdbean against disease powdery mildew caused by
Erysiphe polygoni, where KUP-34 recorded as significantly high in leaf
thickness, phenol content, trichome density, and lowest in total sugars, stomatal size, reducing sugars and non-reducing sugars compared to highly susceptible genotype LBG-623
K e y w o r d s
Black gram,
Morphological and
Biochemical and
Erysiphe polygoni
Accepted:
17 June 2020
Available Online:
10 July 2020
Article Info
Trang 2The objective of the current research was to
check the effect of powdery mildew on
different physio-biochemical traits in resistant
and susceptible pea genotypes
The possibility of these traits viz., electrolyte
leakage, reducing sugars, non-reducing
sugars, total sugars and plant dry weight as
selection criteria for powdery mildew
resistance was also assessed
Materials and Methods
The experiment was conducted during rabi
2015-16, Agricultural College Farm and
Department of Plant Pathology, Agricultural
College, Bapatla, Guntur District
Geographically the Agricultural College
Farm, Bapatla is situated at an altitude of 5 m
above the mean sea level and at 800 30′ E
Longitude and 150 54′ N Latitude and seven
km away from the coast of Bay of Bengal
Morphological Characters of Selected
Blackgram Genotypes
Estimation of Leaf Thickness (mm)
A total of 16 genotypes indicating all
categories of reactions were selected from the
genotypes evaluated during kharif
2015-16,(data not shown) and were sown in three
replications of 5 m each at Agricultural
college, Bapatla, Department of plant
pathology during rabi 2015-16 Three plants
were selected at random from each genotype
at 45 DAS
Three leaves from each plant were selected
randomly from each plant to measure leaf
thickness by using Venier callipers (Perez et
al., 2013) Same genotypes and similar
sampling method have been used for
estimation of stomatal frequency, trichome
density, and biochemical analysis
(number of stomata/mm 2 )
Healthy trifoliate leaf of from 45 days old have been collected and then were smeared with synthetic gum It’s gum is being allowed
to dry, flakes have been peeled and mounted
on microscope glass slide Number of stomata per mm2 was counted using ocular micrometer with 40 X objective lens
(Varadarajan and Wilson, 1973)
Estimation of Trichome Density (5 mm dia leaf disc)
Circular leaf discs with such a diameter 5 mm have been made from punching machine soaked in saffron dye for 5-10 min which were used to validate the number of hairs using stereo zoom microscope as outlined by
Tagger and Gill (2012)
Biochemical Analysis Glassware and sterilization
The glassware used in this study viz., Borosil,
Petri plates, conical flasks, test tubes, pipettes, measuring cylinders, beakers, micropipettes and microtips have been used in this study Glassware was washed first with detergent powder and then rinsed under tap water Subsequently they was kept overnight in cleaning solution prepared by mixing 75 g of potassium dichromate, 500 ml of concentrated sulphuric acid and 1000 ml of distilled water and rinsed with tap water followed by distilled water and analytical or reagent grade chemicals were used in the latter study
Estimation of Total phenols
Total phenols were estimated by Folin-Ciocalteau Reagent method (Malick and Singh, 1980)
Trang 3Estimation of total sugars
Total sugars were estimated following
Anthrone method (Hodge and Hofreiter,
1962)
Estimation of reducing sugars
Reducing sugars estimation was carried out
by Dinitrosalicylic acid method (Miller,
1959)
Estimation of non-reducing sugars
The non-reducing sugar quantity was
determined by deducting the reducing sugar
content from that of the total soluble sugars
Statistical Analysis
The data obtained from all the experiments
were statistically analyzed following the
standard procedures (Gomez and Gomez,
1984)
Morphological characters was measured by
concerned apparatus as mentioned above, as
well as biochemical analysis measured by
spectrophotometer and estimated according
their formulas
Results and Discussion
Among morphological characters, features of
stomata, cuticle and trichome morphology can
impact disease resistance (Niks and Rubiales,
2002)
Morphological and Vegetative Characters
in Selected Blackgram Genotypes
Leaf thickness (µm)
Significantly highest leaf thickness was
observed in highly resistant genotypes
KUP-34 (201.4 µm), KUP-40 (191.3 µm) and four
moderately resistant genotypes KUP-12 (176.2 µm), KUP-6 (173.6 µm), KUP-11 (171.7 µm), KUP-31 (167.3 µm) which were
on a par
Two susceptible genotypes KUP-39 (134.8 µm), KUP-25 (135.7 µm) and four
moderately susceptible genotypes viz.,
KUP-27 (138.1 µm), KUP-30 (139.9 µm), KUP-4 (143.1 µm) and KUP-5 (141.8 µm) showed significantly less leaf thickness compared to highly resistant category and were on par Highly susceptible genotype LBG-623 (107.3 µm) which showed lowest leaf thickness which was on par with one susceptible genotype KUP-37 (118.6 µm) (Table.1)
High degree of resistance and moderate resistance to powdery mildew in the blackgram genotypes can be attributed to higher leaf thickness Cuticle thickness in phlox found to be more in resistant genotype Texas 4n than susceptible genotype
Oklahoma as reported by Andrew et al.,
(1982) Leaf and cuticular or epidermal thickness have also been associated with
powdery mildew resistance (Commenil et al.,
1997)
Stomatal frequency (per mm 2 )
Significantly lowest stomatal frequency was observed in highly resistant genotypes
KUP-34 (88.64/mm2), KUP-40 (99.24/mm2) and were on a par with moderately resistant genotypes KUP-12 (104.55/mm2), KUP-6 (107.58/mm2), KUP-11 (106.82/mm2),
KUP-31 (109.09/mm2) Highly susceptible genotype LBG-623 (193.94/mm2) and one susceptible genotype KUP-37 (184.85/mm2) were found to have highest stomatal frequency and were on a par (Fig 2 and Plate 1) Stomatal frequency was observed to be an important character in determining the resistance in the studied genotypes
Dhanumjayrao et al., (2006) observed high
Trang 4variation in stomatal density in grape
genotypes against powdery mildew
Trichome density (5mm diameter disc)
Significantly higher trichome density was
observed in highly resistant genotypes
KUP-34 (62.33), KUP-40 (59.11) and were on a par
with each other Four moderately resistant
genotypes viz., KUP-12 (42.56), KUP-6
(41.11), KUP-11 (36.00), KUP-31 (38.78)
and two moderately susceptible genotypes
KUP-4 (36.56), KUP-15 (39.44) showed on a
par trichome density with that of resistant
genotypes Rest of the moderately susceptible
genotypes were significantly low trichome
density and were on a par The highly
susceptible genotype LBG-623 (19.33) was
found to possess significantly lowest trichome
density and was on par with one susceptible
genotype KUP-37 (25.0) (Fig 3 and Plate 2)
In highly resistant and moderately resistant
genotypes, the trichome density was found to
be significantly highest which implies that
trichome density can a morphological
character contributing for the resistance of
blackgram genotypes to powdery mildew
Trichomes play an important role by
inhibiting penetration of the pathogen into the
host plant by keeping the pathogen away from
the infection courts (Horsfall and Diamond,
1960) Similarly, Martin and Glover (2007)
found that trichomes can act as physical
barriers to infection High frequency of
trichomes can also prevent mycelial
penetration and infection of other biotrophic
fungi (Shalik, 1985) From the work of
Kortekamp and Zyprian (1999), it appears
that increased number of hydrophobic
pubescences may repel water from leaf
surfaces thus preventing successful
penetration Alternatively, a high trichome
number may simply reduce the frequency of
germ tube contact points that can lead
penetration (Niks and Rubiales, 2002) The results strongly suggested that morphological characters showed resistance to powdery mildew has existed among genotypes
Studies on Biochemical Variability in Selected Black gram Genotypes
Total Phenols content (mg/100 mg)
Significantly difference in phenol content was observed in between two highly resistant genotypes KUP-34 (0.912 mg/100 mg) and KUP-40 (0.861 mg/100 mg) followed by a moderately resistant genotype KUP-12 (0.678 mg/100 mg) Susceptible and highly susceptible genotypes showed lowest phenol content and there are on a par Overall differential phenol contents was observed among highly resistant and moderately resistant genotypes and moderately susceptible and susceptible genotypes (Fig 4) Total phenol content has a role to play in resistance mechanism Concentration of phenolic compounds was usually higher in resistant genotypes than in susceptible genotypes of different crop plants (Arora and
wagle, 1985, Saini et al., 1988) Parashar and
Sindhan (1986) noted higher content of total phenols in stem and leaf of powdery mildew resistant varieties of pea than susceptible Kalia and Sharma (1988) found higher levels
of phenolics and phenol oxidising enzymes in resistant cultivars of pea (P 185 and P 6583) than susceptible, the correlation between the biochemical parameters and disease index
were also high Hattappa et al., (2003) noticed
that the biochemical changes in mulberry
(Morus alba) leaves infected with
Phyllactinia corylea causing powdery mildew
the total chlorophyll, reducing sugar and protein content of mulberry leaves decreased with increased infection by the fungus Helal
et al., (1978) reported that the resistance to E cichoracearum in the cucumber variety
Trang 5Poinsettia was due to a high concentration of
phenols which hindered infection and a low
concentration of sugars prevented
establishment of the pathogen in the host
tissues
Total soluble sugars
Total Sugars content (mg/100 mg)
Significantly lowest sugar content was
observed in highly resistant genotypes
KUP-34 (4.48 mg/100 mg) KUP-40 (4.62 mg/100
mg) and were on a par in their total sugar
content with moderately resistant genotypes
viz., KUP-12 (4.63 mg/100 mg), KUP-6 (4.65
mg/100 mg), KUP-11 (4.66 mg/100 mg) and
KUP-31 (4.74 mg/100 mg) Highest total
sugar content was observed in highly
susceptible genotype LBG-623 (7.39 mg/100
mg) (Fig.5) Resistance of genotypes was
inverse to the total sugar content
Reducing sugars content (mg/100 mg)
Highly resistant genotypes KUP-34 (2.39
mg/100 mg) and KUP-40 (2.36 mg/100 mg)
did not significantly differ in their reducing
sugar content and were on par with
moderately resistant genotypes, KUP -12
(2.70 mg/100 mg), KUP -6 (2.67 mg/100 mg),
KUP -11 (2.67 mg/100 mg) and KUP-31
(2.82 mg/100 mg) in their reducing sugar
contents Moderately susceptible genotypes
viz., KUP-15 (2.89 mg/100 mg) and KUP -18
(2.71 mg/100 mg) observed to have reducing
sugar content on par with highly resistant and
moderately resistant genotypes and one highly
susceptible genotype LBG-623 (4.10 mg/100
mg) were on par in their reducing sugars
content Highly resistant genotypes showed
lowest reducing sugar contents (Fig.6)
Non-reducing sugars (mg/100 mg)
Highly resistant genotypes KUP-34 (1.96
mg/100 mg), KUP-40 (2.26 mg/100 mg)
observed to have lowest non-reducing sugars, they did not differ significantly in their non-reducing sugar content and were on par with
moderately resistant genotypes viz., KUP-12
(1.94 mg/100 mg), KUP-6 (1.64 mg/100 mg), KUP-11 (2.00mg/100 mg) and KUP-31 (1.92 mg/100 mg) in non-reducing sugar content and with highly susceptible genotypes
LBG-623 (3.39 mg/100 mg) for the non-reducing sugar content (Fig.7)
Non reducing sugar content in the genotypes showed an inverse relation with resistance to powdery mildew Early decades, Muhammad
and Ali (2014) found that incidence of
powdery mildew in pea induces changes in reducing sugars, non-reducing sugars, total sugars powdery mildew resistant and
susceptible peas genotypes Dakshayani et al.,
(2005) reported that the susceptible genotypes Chinamung, Pusa Baisakhi and TM-98-50 recorded higher levels of sugars compared to the TARM-18 Parashar and Sindhan (1986) reported that powdery mildew resistant pea varieties (P185 and P388) had higher contents
of total phenols in stem and leaf and low concentration of total sugars and reducing
sugars, than susceptible varieties Gawande et
al., (2002) carried out a biochemical study on
reducing, non-reducing and total sugars and found that resistant genotypes had lower total sugars content before and after infection
Guleria et al., (1997) reported the
post-infection of powdery mildew decrease the reducing sugar content in the leaves of both resistant (DPP68 and JP71) and susceptible cultivars (Bonneville and Lincoln) in pea
Stomatal Frequency and Trichome Density
Braun (1987b) revealed that some lines in the mildew-susceptible germplasm of mulberry of which RFS-135, Mother graft, Shrim-5 and Mizuzawa) have a smaller stomatal density, the number of stomata per unit area of leaf surface and stomatal index were positively
Trang 6correlated with powdery mildew resistance
Eighty per cent of the resistant germplasm
were characterized by high trichome densities
and a high stomatal density and stomatal
index There are some significant genotypic
effects of stomatal frequency on penetration
by powdery mildew pathogens (Lima et al.,
2010) Chattopadhyay et al., ( 2011) evaluated
30 lines of mulberry with contrasting
susceptibilities to powdery mildew (15
resistant and 15 susceptible), susceptible
genotypes showed significant more stomatal
index, stomatal area and less trichome
density Whereas, resistant group was distinguished by 17.4 % lower stomatal density, 12.5% smaller stomatal index per unit leaf area, 20.0 % greater trichome density and 18.0% higher stomatal area compared with the susceptible group Trichome density was negatively correlated with disease severity index and with the accumulative area under disease progression curves (AUPDC)
Georgiev et al., (2013) found positive relation
between the degree of pubescence and resistance to powdery mildew under natural conditions
Table.1 Biochemical characters in selected blackgram genotypes
reaction
Total phenols (mg/100mg)
Total sugars (mg/100mg)
Reducing sugars (mg/100mg)
Non-reducing sugars (mg/100mg)
* In the column means followed by common letter are not significantly different at 5% level by DMRT
Fig.1 Variation of leaf thickness (µm) in selected blackgram genotypes
Trang 7Fig.2 Variation in stomatal frequency (no of stomata mm2) in selected blackgram genotypes
Fig.3 Variation of trichome density (5mm dia leaf disc) in selected black gram genotypes
Fig.4 Variation in total phenols (mg/100 mg) on selected blackgram genotypes
0 0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Blackgram genotypes
Trang 8Fig.5 Variation in total sugars (mg/100 mg) on selected blackgram genotype
Fig.6 Variation of reducing sugars (mg/100 mg) in selected blackgram genotypes
Fig.7 Variation of non-reducing sugars (mg/100 mg) in selected blackgram genotypes
Trang 9Plate.1 Variation in stomatal frequency in powdery mildew disease resistant (KUP-34) and
susceptible (LBG-623) black gram genotypes
Plate.2 Variation in trichome density in powdery mildew disease resistant (KUP-34) and
susceptible (LBG-623) black gram genotypes
Resistance
Involvement of phenolic compounds in many
aspects of plant parasite relationship other
than plant protection has been reported by
Friend in 1979 The role of phenolics in the
resistance mechanisms in plants has been
reviewed by several workers (Allen, 1959;
Agrios, 2005) Concentration of phenolic
compounds was usually higher in resistant
genotypes than in susceptible genotypes of
different crop plants (Arora and Wagle, 1985
and Saini et al., 1988).Mandahar and Garg
(1975) observed that okra leaves infected with
powdery mildew (E cichoracearum) had
higher reducing sugars content than healthy
leaves Helal et al., (1978) reported that the resistance to E cichoracearum in the
cucumber variety Poinsettia was due to a high concentration of phenols which hindered infection and a low concentration of sugars prevented establishment of the pathogen in
the host tissues Guleria et al., (1997) reported
the post-infection decrease the reducing sugar content in the leaves of both resistant (DPP68 and JP71) and susceptible cultivars (Bonneville and Lincoln) of pea against powdery mildew Sridhan and Parashar (1984) found higher content of total phenols, O-dihydric phenols, P, K, Zn, and Cu but lower of N, Mn, and Fe in the foliage of resistant and moderately resistant varieties of pea compared to susceptible Parashar and
Trang 10Sindhan (1986) noted higher content of total
phenols and Orthodihydro-phenols and lower
of total sugars and reducing sugars in stem
and leaf of powdery mildew of resistant
varieties of pea than susceptible Kalia and
Sharma (1988) found higher levels of
phenolics and phenol oxidising enzymes in
resistant cultivars of pea (P 185 and P 6583)
than susceptible cultivars, the correlation
between the biochemical parameters and
disease index were also high Sharma et al.,
(1996) found higher total phenol content in
powdery mildew resistant varieties of pea viz.,
JP 179, JM 5, PMR 3 AND PMR 4
Gawande et al., (2002) carried out
biochemical study on reducing, non-reducing
and total sugars and total phenols before and
after powdery mildew infection in seven
mungbean genotypes found that resistant
genotypes had higher total phenols before and
after infection The total phenols were
positively correlated with resistance
Whereas, sugars were negatively associated
with disease resistance Dakshayani et al.,
(2005) reported that the susceptible genotypes
Chinamung, Pusa Baisakhi and TM-98-50
recorded higher levels of sugars compared to
the TARM-18 Muhammad and Ali (2014)
found that incidence of powdery mildew in
pea induces changes in reducing sugars,
non-reducing sugars, total sugars powdery mildew
resistant and susceptibility of peas genotypes
In conclusion the 15 blackgram genotypes,
significantly highest leaf thickness was
observed in highly resistant genotypes
KUP-34 (201.4 µm), KUP-40 (191.3 µm) Highly
susceptible genotype LBG-623 (107.3 µm)
which showed lowest leaf thickness which
was on par with one susceptible genotype
KUP-37 (118.6 µm) Significantly lowest
stomatal frequency was observed in highly
resistant genotypes KUP-34 (88.64/mm2),
KUP-40 (99.24/mm2) Highly susceptible
genotype LBG-623 (193.94/mm2) and one
susceptible genotype KUP-37 (184.85/mm2) were found to have highest stomatal frequency Significantly higher trichome density was observed in highly resistant genotypes KUP-34 (62.33), KUP-40 (59.11) and were on a par with each other The highly susceptible genotype LBG-623 (19.33) was found to possess significantly lowest trichome density Significantly higher phenol content was observed in highly resistant genotypes KUP-34 (0.912 mg/100 mg) and KUP-40 (0.861 mg/100 mg) and one moderately resistant genotype KUP-12 (0.678 mg/100 mg) Highly susceptible genotype LBG-623 recorded the lowest total phenol content (0.299 mg/100 mg)
Significantly lowest sugar content was observed in highly resistant genotypes
KUP-34 (4.48 mg/100 mg) KUP-40 (4.62 mg/100 mg) and were on a par Highest total sugar content was observed in highly susceptible genotype LBG-623 (7.39 mg/100 mg) Highly resistant genotypes KUP-34 (2.39 mg/100 mg) and KUP-40 (2.36 mg/100 mg) showed
lowest reducing sugars and there on a par
Highly resistant genotypes KUP-34 (1.96 mg/100 mg), KUP-40 (2.26 mg/100 mg) showed observed to have lowest non reducing sugars, they did not differ significantly in their non-reducing sugar content
Acknowledgement
Authors are grateful to Head, Department of Plant Pathology, Regional Agricultural Research Station, Lam, Guntur District, Agricultural College Farm for providing the necessary facilities to undertake this work
References
Agrios, G.N 2005 Powdery mildews Plant
Pathology, 5thed San Diego USA Elsevier Academic Press 346
Allen, P.J 1959 Physiology and biochemistry